EP1303349A1

EP1303349A1 - Method and device for producing biopolymer arrays

Info

Publication number: EP1303349A1
Application number: EP01940302A
Authority: EP
Inventors: Heinz Eipel; Stefan Matysiak
Original assignee: Deutsches Krebsforschungszentrum DKFZ; BASF SE
Current assignee: Deutsches Krebsforschungszentrum DKFZ; BASF SE
Priority date: 2000-04-06
Filing date: 2001-04-06
Publication date: 2003-04-23
Also published as: KR20020097216A; RU2290259C2; NO20024711D0; NO20024711L; US20030143316A1; CZ20023316A3; CN1422175A; AU2001273927A1; WO2001076732A1; CA2405160A1; JP2003530548A; IL152050A0; DE10017105A1; RU2002129601A; IL152050A; CN1301796C

Abstract

The invention relates to a method and device for producing biopolymer arrays (15) on supporting substrates (4, 14), whereby the biopolymers to be applied can be withdrawn from one or more different biopolymer stores. According to the invention, a capillary tip (1) of a capillary tube (2) that can be multidimensionally displaced is controlled for transferring the smallest amounts of liquid to substrate surfaces (14) via a miniature valve (5) provided for filling and via a miniature valve (7) provided for rinsing the capillary tube (2).

Description

Method and device for producing biopolymer fel

The invention relates to a method and a device for producing biopolymer fields (arrays), nucleic acids, proteins and / or polysaccharides for arranging sample quantities of these substances on a carrier or a carrier material.

For the highly parallel analysis of biopolymers - for example nucleic acids, proteins and / or polysaccharides - arrangements of many small sample quantities in drop form are generally applied to flat carriers or carrier substances. Plastic foils are used as carriers for the sample quantities to be applied, or membranes or specimen slides, as are often used in microscopy. In typical analysis applications, several hundred to several thousand analysis spots are applied to a carrier.

To apply the extremely small amounts of liquid of the samples to be analyzed in the range from a few picoliters to a few nanoliters on supports or carrier materials, use is made, for example, of the inkjet printing technique. In the case of the ink-spray Drackcrmik, the sample quantities of the liquids to be analyzed are exposed to greater mechanical and / or thermal loads, which can impair the sensitive biopolymers. With this application technique, undesired formation of gas bubbles can also frequently occur, which result in the exact discharge of the liquid drops and thus a regularly arranged one Hinder analysis field. Furthermore, disturbances can often occur due to the fact that the viscosities of the quantities of liquid to be applied are very different.

A method is known from Science 270, 1995, pp. 467-470, M. Schena et al., Which is based on the fountain pen method. In this solution known from the prior art, metal pins are provided with a molded tip. These are immersed in the liquid to be pipetted; part of the liquid to be applied remains on the surface of the pen tip; this later reaches the surface of the carrier or the carrier material to be loaded when the pen tip is lowered. A disadvantage of this technique is the limited liquid absorption capacity of the deformed pen tip if, after the liquid absorption, many support surfaces are to be dabbed with the same pattern to form arrays to be analyzed.

If furrows or slots are provided on the metal pin tips dipping into the sample containers in order to increase the absorption capacity of the liquid to be applied, then these have the disadvantage of difficult and lengthy cleaning. However, cleaning is imperative in order to avoid carryover of sample substances if the metal pen tip sinks into a container with a new type of sample and remnants of the previously applied substrate still adhere to the tip, so that the new sample spot on the substrate does not contain substances the previously transferred stain is contaminated.

In view of the disadvantages of the solutions known from the prior art, the object of the present invention is to arrange biopolymer fields or arrays to be analyzed inexpensively and reliably using simple means. According to the invention, this object is achieved in that, in a method for producing biopolymer surfaces on carrier substrates, the biopolymers to be applied being taken from one or more sample stocks, a capillary tube of a capillary tube which can be moved in a multidimensional manner for transferring the smallest amounts of liquid to substrate surfaces via a miniature valve serving for filling and can be controlled by another miniature valve used for flushing.

The advantages of this solution can be seen primarily in the fact that the method proposed according to the invention can be used to easily load a large number of carrier substance platelets with a single filling of the capillary. To avoid sample carry-over, two rinse cycles on the capillary have proven to be sufficient so that mutual contamination of the sample stocks and the transferred samples can be sufficiently ruled out for practical use. On the other hand, the rinsing of the capillary in each case holding the sample quantity can be repeated as often as desired by the two independently controllable miniature valves.

In a further embodiment of the method on which the invention is based, a number of capillary tubes can be connected to the miniature valves. This enables a parallel application of several smallest amounts of liquid to the surface of a substrate or a substrate material.

If several capillary tubes are used at a distance from one another, a larger number of liquid samples to be analyzed can be applied simultaneously by processing several carrier surfaces in parallel. According to a further advantageous development of the idea on which the invention is based, the plurality of capillary tubes can be arranged with respect to one another in such a way that their distance from one another corresponds to the distances between two sample quantities of biopolymer substances with which these are applied to the surface of the carrier substrate.

The more regular the arrangement of the smallest amounts of liquid to be analyzed is on the surface of the substrate carrier, the more precisely an evaluation of the applied liquid samples can be carried out and the sooner a downstream analysis method can be automated.

In a preferred embodiment of the method proposed according to the invention, the one or more capillary tubes can be moved in the X or Y direction, and an immersion movement in the Z direction can also be carried out to receive a liquid supply from a substrate container. Due to the controllability of the respective capillary tubes in the three coordinate directions, a maximum utilization of the space on analysis platelets can be achieved. To control and move the one or more capillary tubes that apply the smallest amounts of liquid to be analyzed to the respective carrier surfaces, a commercially available, computer-supported plotter that can be moved in the X and Y directions is advantageously used. By controlling a commercially available plotter by means of a personal computer (PC), inexpensive movement and reliable control of the one or more capillary tubes can be achieved.

Instead of a commercially available plotter with which the one or more capillary tubes can be moved in the X direction or the Y direction, computer-assisted positioning tables can also be used. According to the invention, a device for generating biopolymer fields on carrier substrates is also proposed, the biopolymers to be applied being able to be taken from one or more different sample stores, a multidimensionally movable glass capillary tip of a capillary tube for transferring minute amounts of liquid to substrate surfaces via a miniature valve serving for filling and via a flushing device the capillary serving miniature valve can be controlled. In a further embodiment of the device for producing biopolymer fields proposed according to the invention, the capillary tips are drawn out to the smallest liquid-absorbing ends with an outside diameter in the range between 10 μm and 1000 μm. In a particularly preferred embodiment, the capillary tips are designed with an outer diameter of 50 μm to 300 μm at the end that receives the smallest amounts of liquid.

The control of the one or more capillary tubes can be carried out by a computer-aided plotter, which produces a movement of the capillary tube or tubes in the X or Y direction as well as an immersion movement of the capillary tube together with the liquid supply accommodated therein in the Z direction by the smallest amounts of liquid to be applied to the surfaces of supports or support materials. In an embodiment variant proposed according to the invention, the miniature valves that are provided in the line system to the capillary tube can be designed as pinch valves. These can in particular be provided to support the flexible hose line by a fixed stop, in contrast to which a flexible stop is provided, with which the cross section of the flexible hose line can be closed. The original cross-section of the flexible supply line is restored automatically due to the elasticity of the hose material. The invention is explained in more detail below with reference to the drawing, which comprises a single figure.

The single figure shows a device for carrying out the proposed method according to the invention, in which the capillary tube together with the capillary tube tip can be moved in three directions.

From the illustration according to the single figure, a capillary tube 2 - preferably consisting of glass - emerges, which serves to hold a biopolymer solution to be pipetted. This is immersed in a sample quantity container 3, also referred to as a microtiter plate pot. The opening of the first miniature valve 5 - in the form of a pinch valve, for example - to the atmosphere 6 brings about a pressure equalization with the atmosphere 6, so that a sample quantity 13 rises into the interior of the capillary tube 2 due to the capillary action by the capillary tip 1.

In a preferred embodiment, the capillary tube 2 is made of glass, the outer diameter of the capillary tip is in the diameter range between 10 μm and 1000 μm, in particularly preferred embodiments of the capillary tube proposed according to the invention between 50 μm and 300 μm. In order to take up the biopolymer solution samples to be applied to the surfaces 14 of carrier material 4, the capillary tip 1 of the capillary tube 2 is immersed in the initial solution contained in the container 3. The reference solutions can be located, for example, in pots 3 of a microtiter plate, which can hold 96 or 384 or even 1536 individual samples. When the capillary tip 1 is immersed in the template solution, the valve 1, which controls the supply of a gas flow into the capillary tube 2, initially remains closed. The valve 5, on the other hand, which is received on the T-piece 11 with the flexible feed line 19 to the capillary tube 2, is opened and thus represents a pressure compensation to the surrounding atmosphere 6. a liquid supply 13 moves out of the potty 3 of the microtiter plate, into which the capillary tip 1 has just been immersed, into the interior of the capillary tube 2.

The capillary tip 1 is then pulled out of the stock solution, then positioned in the X and Y directions and moved over the surface 14 of a carrier 4, to which the individual liquid samples to be analyzed are then applied in a biopolymer pattern 15 while maintaining precisely defined distances 16 from one another. When the capillary tip 1 is lowered in the direction 12 (Z direction) toward the surface 14 of the carrier 4, the position of the first valve 5 and the position of the second valve 7 are not changed. By a control device 20, which causes the capillary tube 2 to move in the X direction, Y direction and Z direction; With the interposition of a commercially available plotter, the capillary tips 1 can be lifted off the surface 14 of the carrier material 4 again in a very simple and cost-effective manner, a small spot of biopolymer solution remaining on the surface 14 of the carrier material 4. A suitable control 20 of a plotter used, for example, allows the capillary tube 2 and the liquid supply 13 contained therein to be moved in the X and Y directions according to the control of the plotter, so that successively further carrier surfaces 14 of carrier material 4 are provided with biopolymer stains in the same way can be. The biopolymer stains are preferably applied in a regular pattern 15, the biopolymer patterns preferably being characterized in that the individual sample stains are at a uniform distance 16 from one another.

Before taking a new sample, i.e. before diving into a new one

The capillary 1 must be cleaned thoroughly to avoid sample carry-over. For this purpose, the capillary tip 1 is first moved over a waste container 9; then the first valve 5, one Representing the connection to the atmosphere 6, closed and through the second miniature valve 7 a gas stream, preferably filtered air or nitrogen, let into the interior of the capillary tube 2 via the flexible feed line 19.

For thorough washing, the capillary tip 1 is now moved over a washing vessel 10, whereby after closing the second miniature valve 7, i.e. the gas valve and opening the first miniature valve 5, i.e. of the outside air valve, the capillary tip 1 is lowered into the washing liquid. Due to the onset of capillary force, the washing liquid now flows into the interior of the capillary tube 2. The capillary tip 1 of the capillary tube 2 is then moved again over the waste container 9 and the washing liquid is expelled to the atmosphere 6 by opening the second miniature valve 7 and closing the first miniature valve 5. Alternatively, this can also be done while in the immersed state in the washing liquid if it is ensured that the washing liquid in the washing vessel 10 is continuously renewed, for example by continuous pumping. For this purpose, a pump circuit 17 for the washing fluid can be assigned to the washing vessel 10, in which, on the one hand, new, unused washing fluid can be fed to the washing vessel 10, and on the other hand, already used washing fluid or deposited particles on the bottom of the washing vessel are continuously removed.

The absorption and ejection of washing fluid from the inside of the capillary tube 2 can be carried out as often as desired by appropriately actuating the two miniature valves 5 and 1, which are preferably designed as pinch valves, until the inside of the capillary tube 2 and its outside are sufficiently cleaned and then a continuation of the application of biopolymer arrays to the top side 14 of the carrier substrates 4 to be loaded can take place. The structure of the apparatus shown in FIG. 1 will be described in more detail below with reference to a sample. On the cart of a standard plotter that can be driven in the X and Y directions (for example ROLAND DXY 1150A) there is a small carrier for two miniature pinch valves attached. From a glass micropipette 2, for example a borosilicate glass capillary from Hilgenberg, outer diameter 1.0 mm, inner diameter 0.8 mm, a tip 1 with an outer diameter of approximately 200 μm was drawn out in a gas flame. The outer diameter of the glass pipette 2 (1 mm) fits commonly, but with a sufficiently small amount of play into the stainless steel cannula of a 1.5 x 100 syringe. This cannula can be easily moved as a guide element on the spring holder of an X or Y direction attach commercially available plotters. The glass micropipette 2 is easily movable in the vertical direction in this guide cannula and is not pressed down by the flexible tube 19. Alternatively, this force can be supported by a small spring.

With the commands "pen-up" and "pen-down" of the plotter, controlled via a commercially available PC, the filling element, which holds the capillary tube 2, can be moved up and down. The connection to the capillary tube 2 is established via the T-connector 11 provided in the supply line from the valves 5, 7 to the flexible hose 19.

It has surprisingly been found that with this arrangement, as many carrier plates 4 as can be accommodated on the DIN A3 working area of the plotter used in addition to the master microtite latte, can be charged with a single filling of the interior of the capillary tube 2 with a liquid supply 13. In the production of carriers 4 with biopolymer patterns 15 made of nucleic acid, it has been shown that two washing steps in a solution of 0.5% TWEEN-80 are normally sufficient to rule out sample carryover which is disruptive in practice. It is when cleaning the

Glass capillary 2 to ensure that the capillary tip 1 on the inside of

Washing fluid is wetted, which can be expelled again from the interior of the glass capillary via the gas flow to be applied, controllable through the second mechanical valve 1. By immersing the capillary tip 1 made of glass in a

Vessel containing washing fluid ensures that the outside of the Capillary tip 1 comes into contact with the washing fluid and is thus cleaned of residues of the previously analyzed sample. When blowing out the washing fluid in the immersed state of the capillary tube 2, it should be noted that the outside of the capillary of the capillary tube 2 is also thoroughly washed by the formation of bubbles in the washing solution with the bubble rising on the capillary 2.

With the proposed arrangement, an enormous economic advantage can be seen in comparison to previously customary loading arrangements. On the one hand, the availability of very precisely related commercially available capillary tubes 2 plays a role in comparison to the production of precisely ground and specially shaped metal pens, on the other hand, X-Y plotters can be procured very inexpensively as automatically controllable positioning tables and in a system according to the invention for producing biopolymer arrays Integrate on surfaces of supports.

LIST OF REFERENCE NUMBERS

1 capillary tip

2 capillary tubes

3 substrate containers

4 carriers

5 first miniature valve

6 atmosphere

7 second miniature valve

8 Gas flow supply line

9 waste container

10 washing vessel

11 T connector

12 Travel Z direction towards capillary tube 2

13 recorded sample

14 carrier surface

15 biopolymer patterns

16 distance

17.1 Washing fluid supply

17.2 Wash fluid drain

18 washing fluid level

19 flexible supply line

20 control device

X-direction

Y-direction

Z direction (application direction)

Claims

claims

1. A method for producing biopolymer fields (15) on surfaces (14) of carrier substrates (4), the biopolymers to be applied being removed from one or more different sample stocks (3), characterized in that a capillary tip which can be moved in several dimensions (1) a capillary tube (2) for transferring the smallest amounts of liquid

Substrate surfaces (14) are controlled via a miniature valve (5) serving for filling and via a miniature valve (7) serving for flushing the capillary tube (2).

2. The method according to claim 1, characterized in that several capillary tubes (2) to the miniature valves (5), (7) are connected.

3. The method according to claim 2, characterized in that the plurality of capillary tubes (2) are operated in parallel.

4. The method according to claim 2, characterized in that the distance at which the plurality of capillary tubes (2) are arranged to each other correspond to the distance between the storage vessels (3) to each other on a template plate.

5. The method according to claims 1 and / or 2, characterized in that the one or more capillary tubes (2) are movable in the X, Y direction and an immersion movement (12) in the Z direction for receiving a liquid supply (13) export from the sample container (3).

6. The method according to claims 1 and / or 2, characterized in that a one or more capillary tubes (2) in the X direction and Y direction are used for the method, a commercially available computer-supported plotter.

7. The method according to claims 1 and / or 2, characterized in that for moving the one or more capillary tubes (2) in the X direction or X direction, a computer-assisted positioning table is used.

8. Apparatus for producing biopolymer fields (15) on surfaces (14) of carrier substrates (4), the biopolymers to be applied being taken from one or more different sample stores (3), characterized in that one or more multi-dimensionally movable glass capillary tubes (2 ) with a capillary tip (1) for transferring the smallest amounts of liquid to substrate surfaces (14) via a first miniature valve (5) for filling and a miniature valve (7) for flushing the capillary tube (2).

9. The device according spoke 8, characterized in that the capillary tips (1) at the liquid-absorbing end in one

Outside diameter is extended in a range between 10 microns and 1000 microns.

10. The device according to claim 9, characterized in that the capillary tube tip (1) at the liquid-absorbing end

Has outside diameter between 50 microns and 300 microns.

11. The device according to claim 8, characterized in that the control of one or more capillary tubes (2) is carried out by a computer-aided X-, Y-plotter, which is a method of the or the capillary tube (2) in X-

Direction and / or Y direction effects.

12. The device according spoke 8, characterized in that the miniature valves (5), (7) are designed as hose pinch valves.

13. The device according spoke 12, characterized in that the hose pinch valves (5), (7) as a flexible feed line (19) to the glass capillary (2) surrounding stops, one of which is fixed relative to the flexible hose line (19) and the other is movable in relation to the fixed stop for narrowing the cross section to bring about a closure in the flexible hose line (19).